Acidification of a wide array of intraellular organelles, including clathrin- coated vesicles, lysosomes, chromaffin granules, and other endomembranes, has been shown to be mediated by a new class of electrogenic, proton translocating ATPases. In addition, renal distal nephron acidification is effected by a similar, if not identical proton pump. It is the central aim of this proposal to define the molecular characteristics of a prototype of this class of H+ ATPases, the clathrin-coated vesicle proton pump. As has been recently demonstrated, this proton pump is a 530 kDa complex composed of a maximum of 8 polypeptides. Several key issues are detailed in this proposal including further purification of the enzyme in a reconstitutively- active form, identification of the function of demonstrated subunits of the holoenzyme, and identification of differences among the proton pumps of various organelles which might account for their disparate localizations and functions. Further purification will be attempted using immunoaffinity and nucleotide affinity chromatography. Assessment of purification will be based upon determination of the minimal polypeptide (subunit) requirements for reconstitution of the isolated ATPase into liposomes prepared from defined lipids. Analysis of subunit function will be achieved by the dissociation and reassembly of subunits of the proton pump, and by analysis of a group of Chinese hamster ovarian cells which carry temperature sensitive mutations in vacular acidification. Delineation of the molecular characteristics of the clathrin- coated vesicle proton pump will provide the groundwork necessary for future studies of the molecular biology, and membrane biogenesis of this multimeric enzyme.